SUMMARY Decades of research have led to a detailed mechanistic understanding of bacterial translation initiation. Almost all this work has focused on ?canonical? translation initiation, which involves association of the 16S ribosomal RNA with the mRNA at a Shine-Dalgarno (SD) sequence. Our studies, along with very recent publications, have uncovered a surprisingly large class of non-canonical bacterial mRNAs: leaderless mRNAs (LLmRNAs) lack a 5?-untranslated region (UTR) and the critical SD sequence. LLmRNAs are rare in E. coli and have received little attention. However, our published and preliminary data indicate that leaderless translation initiation is both common and robust in mycobacteria (~25% of all mRNAs). The surprising discovery that leaderless-pervasive bacteria are far more common than once thought identifies a critical gap in our understanding of ribosome biology and translation in bacteria and, therefore, is of fundamental biological significance. Ribosomes are major antibiotic targets, but leaderless translation represents a new essential process for which novel inhibitors could be identified and developed as anti-mycobacterial agents. The goal of this proposal is to determine the mechanism by which ribosomes recognize, bind and initiate translation on an LLmRNA in the absence of a 5?UTR. Our preliminary data suggest that leaderless translation is achieved by an initiation complex that includes a 70S ribosome bound to mycobacterial RNA polymerase at the site of transcription initiation, suggesting that nascent mRNA feeds directly into the ribosome, obviating the need for a SD sequence. While co-transcription and translation is an established paradigm for bacteria, it has only been considered in the context of the elongating complex on a leadered mRNA. Our data support a simple model of co-initiation of transcription and translation that allows efficient translation of LLmRNAs found in bacteria, archaea and mitochondria. In the proposed work, we aim to identify and characterize initiation complexes that enable leaderless translation in mycobacteria. M. smegmatis is ideal for the proposed work because it is genetically tractable, which will facilitate the first mechanistic study of LLmRNA translation in a bacterial species that natively expresses large numbers of leaderless transcripts. Our collective expertise in mycobacterial ribosome structure and biology has provided a solid foundation for the proposed studies. We have developed or are developing complementary in vitro and in vivo tools and assays that are now set to focus on dissecting leaderless initiation complexes, to catch up with the well-described models of canonical leadered translation initiation. Our potent proposed combination of traditional in vitro biochemistry with new initiation complex inhibitors and in vivo ribosome profiling will promote a deeper understanding of the complex process of leaderless gene expression in mycobacteria.